ESCRT disruption provides evidence against signaling functions for synaptic exosomes

  1. Erica C Dresselhaus
  2. Kathryn P Harris
  3. Cassandra R R Blanchette
  4. Kate Koles
  5. Steven J Del Signore
  6. Matthew F Pescosolido
  7. Biljana Ermanoska
  8. Mark Rozencwaig
  9. Rebecca C Soslowsky
  10. Michael J Parisi
  11. Bryan A Stewart
  12. Timothy J Mosca
  13. Avital Adah Rodal

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Abstract

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

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    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary: In this paper, Dresselhaus et al (2023) investigate the possibility that known cargoes of extracellular vesicles (EVs) released at the Drosophila neuromuscular junction have cell-autonomous functions rather than functions specifically conferred as a condition of their release in EVs, in vivo. To do so, authors focus their studies on use of Tsg101-KD, a mutant of the ESCRT-I machinery, of the ESCRT EV biogenesis pathway, and are able to show that for some endogenously-expressed, fluorescently-tagged cargoes, fluorescence intensity in the pre-synaptic compartment is significantly elevated (Syt4 and Evi) and the postsynaptic intensity in the muscle is significantly decreased (Syt4, Evi, APP, and Nrg).

    We note that throughout our study, we detected endogenous Nrg with a well-characterized monoclonal antibody, not a fluorescent tag. We and others previously demonstrated that endogenous Nrg detected by this antibody is trafficked from neurons into EVs, using the same pathways as other EV cargoes such as Syt4, APP and Evi (Blanchette et al., 2022; Enneking et al., 2013; Walsh et al., 2021). Thus, the EV trafficking phenotypes in our study are consistent across fluorescently tagged cargo (endogenous knockin for Syt4 and GAL4/UAS-driven for APP and Evi), as well as for untagged, endogenous Nrg, thus controlling for effects of either overexpression or tagging.

    These findings suggest that these cargoes become trapped in the endosomal system (colocalizing with early, late, and recycling endosomal compartments), rather than undergoing secretion in EVs targeting post-synaptic muscle and glia as usual. This phenotype is recapitulated for select cargoes using mutants of both early and late components of ESCRT pathway machinery. They further characterize the Tsg101 mutant, demonstrating co-occurrence of an autophagic flux defect, but as the cargo phenotype is present without induction of the autophagic flux defect for their Hrs mutants, authors suggest the overlapping role of Tsg101 in autophagy is independent of its role in the ESCRT pathway/ EV secretion. Subsequently, they use previously defined functional phenotypes of the Evi (number of active zones, number of boutons, number of developmentally-arrested ghost boutons) and Syt-4 (number of transient ghost boutons and mEJPs) cargoes to show a minimal dependence on cargo delivery via ESCRT-derived EVs for these cargoes to carry out their synaptic growth and plasticity functions in vivo. However, it should be notes that for Evi/ Wg cargo, there is a slight increase in developmentally-arrested ghost boutons suggesting the cargo may not be entirely independent of EV-mediated cargo delivery. Finally, authors express an anti-GFP proteasome-directed nanobody using motor neuron or muscle-specific drivers and find that Syt4-GFP cargo doesn't enter muscle cytoplasm as fluorescence is maintained and cargo is not degraded by the muscle proteasome. While authors suggest this as evidence of EV-mediated transfer for cargo proteostasis, it is not explicitly shown that Syt4 cargo is, in fact, trafficked and degraded by the lysosome or hypothesized how Syt4 function or post-synaptic localization may be carried out independently of EVs.

    We have added new data showing that Syt4 is taken up by glial and muscle phagocytosis (Fig. 7), and included in the discussion several possible interpretations for how Syt4 activity is carried out independently of its traffic into EVs. Indeed we believe it is more likely to function in the presynaptic neuron rather than the postsynaptic muscle.

    Major comments:

    R1.1 It is difficult to evaluate the findings of this study without knowing the extent of ESCRT pathway impairment. Please provide data quantifying the degree of knockdown/ mutant expression for each ESCRT component (i.e., western blot)

    To address the reviewer’s request to specifically measure the degree of knockdown in the RNAi lines, we tested all available reagents. Unfortunately no Drosophila Tsg101 antibody exists and we did not receive a reply to our requests for a Shrub antibody. An Hrs antibody exists, but we found that none of three available Hrs RNAi lines depleted Hrs signal, or caused a phenotype similar to the HrsD28 point mutant, suggesting that they are not effective at knocking down the protein. Therefore, we were unable to specifically measure the level of depletion in motor neurons for RNAi of Tsg101, Shrub, or Hrs.

    However, we can make a strong argument that our knockdowns were sufficiently effective to answer the questions in our study. We used RNAi as only one of several complementary tools to manipulate ESCRT function (i.e. we also used loss-of-function mutants (HrsD28/Deficiency) and dominant negative mutants (Vps4DN)). These mutants caused a comparable and severe loss of EVs to RNAi (Fig 2): therefore the extent of depletion in the RNAi experiments was sufficient to cause a similarly severe phenotype as genomic or DN mutations, meeting the definition of a bona fide loss-of-function. We also know, since we used these complementary strategies, that the phenotypes we observe are very unlikely to be due to off-target effects of the RNAi.

    More importantly, what is directly relevant for our subsequent functional experiments is to know the extent of EV depletion, which we have explicitly measured throughout the paper. It is unclear what additional insights would be gained by knowing whether the strong Tsg101 and Shrub RNAi phenotypes are due to incomplete versus complete knockdown, given that we do measure the extent of EV depletion under these conditions. Further, we note that tsg101 null mutants die as first instar larvae (Moberg et al., 2005), raising the possibility that a more complete knockdown in neurons would be lethal early in development and make our study impossible. Indeed HrsD28 is an early stop that preserves the VHS and FYVE domains but truncates the C-terminal ⅔ of the protein. Its (occasional) survival to third instar indicates that it may be a severe hypomorph rather than a null.

    We have added a sentence in the text (p12 line 21-25) to clarify that we do not know the exact extent of knockdown for our RNAi experiments, but that by genetic definitions, they meet the criteria of a loss-of-function manipulation.

    R1.2 Loss of ESCRT machinery likely disrupts the release of small EVs to a significant extent; however, the authors do not show that EV release is entirely lost, only that 1) cargoes are backed up in the endosomal system due to endosomal dysfunction and 2) fluorescence of cargoes in the postsynaptic compartment is diminished. To claim that ESCRT-derived EVs with the relevant cargoes are lost, the authors should perform immunogold labelling with TEM. This would provide direct evidence that the cargoes examined here are packaged in ILVs, and that the ILVs are of a size (~50-150nm) consistent with exosomes (which should really be referred to as small extracellular vesicles (sEVs) per the minimal information for studies of extracellular vesicles (MISEV 2018 [https://doi.org/10.1080/20013078.2018.1535750]) Additionally, EM would show the loss of cargo packaging and provide information about where these cargoes localize in the presence of ESCRT mutants/loss-of-function.

    EM (including some limited immunoEM) studies requested by Reviewer 1 have previously been performed in this system by us and by the Budnik and Verstreken labs (Koles et al., 2012; Korkut et al., 2009; Korkut et al., 2013; Lauwers et al., 2018; Walsh et al., 2021). MVBs at the NMJ contain ~50-100 nm ILVs, and can often be seen proximal to or fusing with the plasma membrane. Mutants such as Hsp90 that block this fusion also block EV release, arguing that these MVBs are the source of EV (Lauwers et al., 2018). By immunoEM, the EV cargo Evi localizes to MVBs (Koles et al., 2012). ~50-200 nm structures containing immunogold against Evi were also observed in the subsynaptic reticulum between the neuron and the muscle, as well as in membrane compartments in the muscle cytoplasm (Koles et al., 2012; Korkut et al., 2009). Thus, the criteria requested by the reviewer have previously been established in this system.

    In response to the reviewer’s request to show that these structures are altered in ESCRT mutants, we attempted immunoEM experiments in the Tsg101KD condition. However, similar to the previously published results (Koles et al., 2012; Korkut et al., 2009), immunoEM in thick tissue such as Drosophila larval fillets is quite challenging, and we found it very difficult to retain immunogenicity together with excellent fixation and preservation of membrane structures, such that we could rigorously measure compartment morphology and size. Even if we did achieve good structural preservation, exosomes are ambiguous in complex membrane-rich tissues, since cross-sections through the extensively infolded muscle membrane (e.g. see Fig 3B) are very similar in size to EVs.

    As an alternative and more robust approach, we used STED microscopy, with a resolution of ~50nm, where we could conduct a rigorous and properly powered study of directly labeled EV cargoes (New data in Fig. S1). We show that postsynaptic Nrg and APP-GFP are found in structures with a mean diameter of ~125 nm, consistent with small EVs or exosomes, and these are strongly depleted in the Tsg101KD animals (to similar levels as antibody background far from the site of EV accumulation), as expected. Note that we are able to detect particles significantly smaller than 125 nm in the distribution, suggesting that the resolution of our system is sufficient to measure EV width.

    We also note that several of these cargoes are detected via an intracellular tag (Syt4, APP, Evi) or antibody against an intracellular domain (Nrg), so by topology they must be membrane-bound in the EVs rather than cleaved from the cell surface. We and others have previously shown that this postsynaptic signal is entirely derived from the presynaptic neuron, by using neuronal UAS-expression of a tagged protein, by neuronal RNAi of the endogenous gene, or by the tissue-specific tagging approach in the current manuscript (Fig. S4). We have also previously shown that these puncta contain the tetraspanin Sunglasses (CG12143/Tsp42Ej), which is an EV marker (Walsh et al., 2021). We have added new data to our manuscript (Fig. S1A) to show that neuronally-derived tetraspanin EVs are depleted in upon Tsg101KD. Therefore, the reviewer’s point “2) fluorescence of cargoes in the postsynaptic compartment is diminished.” is the most direct and sensitive test of trans-synaptic cargo transfer, and is the precise parameter that we are trying to manipulate to test the functions of this transfer.

    We believe that light microscopy showing loss of presynaptically-derived cargoes in the postsynaptic region is the best and most direct argument for loss of EV secretion, compared to the ambiguity of EM. It is also exactly the method that led to the proposal for the signaling function of EVs in previous work, which our current manuscript is revisiting. We are now using improved tests of that original hypothesis by examining it in light of additional membrane trafficking mutants (and finding that it no longer holds up). Overall, given the preponderance of evidence from the preceding literature and our studies indicating that (1) these cargoes are indeed in EVs and (2) we see a strong enough depletion of transsynaptic transfer to challenge the hypothesis that EVs serve signaling functions (see R1.3 response below), we are reluctant to spend more time attempting immunoEM which is not likely to resolve membrane structures.

    To address the point of EV terminology used in our manuscript, we think it is very unlikely that the postsynaptic structures are not exosomes. The criteria defined by MISEV for exosomes is that they are endosomally-derived from MVBs, ideally with the EV “caught in the act of release” upon fusion with the plasma membrane. As noted above, cargoes such as Syt4 and Evi are observed by immunoEM in MVBs, and these can be found in the process of fusing with the plasma membrane (i.e. caught in the act of release) (Koles et al., 2012; Korkut et al., 2009; Korkut et al., 2013; Lauwers et al., 2018). Mutants that block MVB fusion also block EV release at the NMJ (Lauwers et al., 2018). These EVs require ESCRT for their formation and are trapped in endosomes rather than the plasma membrane upon ESCRT depletion (this study). They depend on multiple components of the endosomal system (Rab GTPases, retromer) for their formation (Koles et al., 2012; Walsh et al., 2021). Taken together, it seems to us that there is sufficient data to argue that these are exosomes. However, as the reviewers requested, we have called them EVs in the revised paper (and only suggest they are exosomes in the discussion).

    R1.3 Other biogenesis pathways utilize multivesicular bodies to generate EVs, most prominently the nSMase2/ceramide synthesis pathway (which operates in an ESCRT-independent manner). It is possible that this pathway compensates when there are defects in the canonical ESCRT pathway. Thus, it is imperative for the authors to show that the cargo secretion no longer occurs in the presence of ESCRT mutations/loss-of-function. The authors should also use nSMase2 pathway mutants to see if the phenotypes in cargo trafficking (i.e., pre/ post-synaptic protein levels) are recapitulated.

    The reviewer asked us to show that cargo secretion does not occur in the ESCRT mutants. We reiterate that at the limits of detection of our assay, we see a very strong depletion of secretion__, __and that EV cargo levels are not distinguishable from background (Figure S1). Perhaps Reviewer 1’s concern is that since it would never be possible to show that we have depleted EVs completely (i.e. below the level of detection of our assays), that it is not possible to challenge the hypothesis that EV traffic is required for the proposed signaling functions of EVs. Indeed, they mention in their overall assessment “as it is unknown if minor sources of cargo+ EVs are sufficient in maintaining functional phenotype”. We do have some information on this, as described in the manuscript (p3 lines 41-43; p7 lines 25-31; p11 lines 27-30) and as follows: The critical argument against this concern is that other trafficking mutants with residual levels of EVs (rab11 or nwk) do show loss of signaling function (Blanchette et al., 2022; Korkut et al., 2013). Therefore residual EVs, even at the lower level of detection of our assay, are not enough to support signaling. The main difference is that in nwk and rab11 mutants the levels of the cargo in the donor presynaptic neuron are also strongly depleted, unlike in the ESCRT mutants. This strongly suggests that the cargoes are signaling from the presynaptic compartment, rather than in EVs. We have added the nwk mutant to show this baseline in Figure 2A,D. Similarly, our new results showing that hrs mutants retain Wg signaling while Tsg101 mutants do not, despite a similar degree of EV depletion (new data with more cargoes in Figure 2A-F), argues that residual EVs do not account for the lack of disruption of signaling. Finally, we have been transparent in our discussion that trace amounts of EVs could still exist, including by alternative pathways, but are unlikely to provide function (p11 lines 25-33).

    We agree that it might be an interesting future mechanistic direction to ask if the SMase pathway works with or in parallel to the ESCRT pathway (both have been suggested in the literature). However, we do not believe that this is essential for the current work: The SMase pathway is unlikely to be “compensating”, since EVs are already very strongly depleted with ESCRT disruption alone. We also note that SMase depletion may also affect other trafficking pathways (Back et al., 2018; Choezom and Gross, 2022; Niekamp et al., 2022), and therefore might not provide any clarifying information if it did disrupt signaling. In summary, we believe the depletion we see in single ESCRT mutants is sufficient to (1) establish the role of ESCRT in EV traffic in this system, and (2) test the role of transsynaptic transfer in signaling functions of cargoes.

    R1.4 The authors' findings support that cargo trafficking is affected by widespread endosomal dysfunction but doesn't cleanly prove that 1) synaptic sEV release is lost and 2) that cargo-specific sEVs are lost. As previously mentioned, loss of cargo+ ILVs in MVEs by TEM could demonstrate this, but another useful approach would be to include in vitro Drosophila primary neuronal culture/ EV isolation and mass spec/proteomic characterization studies as proof of concept. According to widely agreed upon guidelines in the EV field, the authors should directly characterize their EV population to show 1) the appropriate size distribution associated with exosomes/sEVs, 2) the presence of traditional EV markers (i.e., tetraspanins), 3) changes in overall EV count by ESCRT mutants, and 4) decreased levels of cargo(es) of interest in the presence of ESCRT mutants/loss-of-function. In vitro experiments would be particularly helpful for quantifying the degree of loss of cargo-specific EVs with each ESCRT mutant. These experiments could also investigate the possibility that cargoes are secreted in nSMase2/ Ceramide-derived EVs, by showing that EV cargo levels are unaffected in nSMase mutants.

    Our data already show loss of cargo-specific EVs, defined by puncta of several independent specific cargoes in the extraneuronal space and postsynaptic muscle. To further substantiate this, we have directly characterized our EV population and shown a distribution of ~125 nm extraneuronal structures containing the transmembrane cargoes Nrg and APP (by STED) as well as Evi, Syt4 and the EV marker tetraspanin (by confocal microscopy). This addresses the (1) size distribution, (2) EV marker and (3) count criteria. All these markers (cargoes and tetraspanins) are severely depleted from the postsynaptic area in the ESCRT mutants, satisfying the (4) decreased levels criteria. As noted above, we and others have repeatedly demonstrated that these postsynaptic puncta are derived from neurons, and since we are detecting the intracellular domain in all cases, must be membrane-bound. Others have previously shown by EM that several of these markers are surrounded by membrane and derived from neuronal MVBs (see R1.2). Note that we do not believe that ESCRT mutants must necessarily cleanly show enlarged endosomes without ILVs or a class E vps compartment - instead stalled endosomes appear to be targeted for autophagy in heterogeneous intermediates (Fig 3).

    We do not believe that turning to a heterologous system (e.g. cultured primary Drosophila neurons, which do not even form functional synapses) is usefully translatable to results in neurons in vivo. Data from our lab and many other systems has shown that EV biogenesis and release pathways are highly cell-type specific (p9 lines 8-12), and also differ in different regions of neurons (eg synapses vs soma) (Blanchette and Rodal, 2020). Further, keeping the experimental setup of the original for EV signaling hypothesis is a prerequisite for our improved tests of this hypothesis. We do note that APP, Evi and Syt4 have been demonstrated by us and others to be released from Drosophila S2 cells in EVs defined by differential centrifugation, sucrose gradient buoyancy, electron microscopy and mass spectrometry (Koles et al., 2012; Korkut et al., 2009; Korkut et al., 2013; Walsh et al., 2021). However even if we did measure the precise change in EV number and cargoes upon ESCRT manipulation in these heterologous cells, it would not allow us to conclude that the same quantitative change was happening in the motor neurons of interest in vivo, which is the information we need to conduct our tests of cargo signaling function. All we would learn is whether ESCRT was required in that cell type, which would not be informative for our study.

    We appreciate that EV researchers working in cell culture systems often use a set of approaches including bulk isolation, EM, and mass spectrometry. Our system does not allow for these approaches, but provides complementary strengths of single EV characterization, in vivo relevance with functional assays, and a wealth of genetic tools. MISEV itself states that it does not provide a set of agreed-upon rules that can be applied generically to any experiment. We agree with the MISEV statement that we should use the best available assays for the system under investigation.

    R1.5 During functional tests of Evi+ motor neurons lacking generation of Evi+ EVs, there is a slight defect observed, namely the increased formation of developmentally arrested ghost boutons when Evi secretion in sEVs is lost. As mentioned, Evi is a transporter of Wg and it is possible for Wg to be transmitted between cells via normal diffusion. Thus, some basal levels of Wg may be reaching the muscle when its transfer via sEVs is abolished, and these basal levels may be sufficient to phenocopy the WT in the number of active zones and boutons. Is it possible that this element of Evi/ Wg function is dose-dependent and thus reliant on the extra Evi/ Wg transferred via sEVs? If possible, the authors should use a Wnt-signaling pathway reporter (i.e., fluorescently tagged Beta-Catenin) to measure the levels of Wnt signaling activity in the muscle when Evi/Wg+ EVs are present vs. abolished. If the degree of Wnt signaling (readout would be intensity of fluorescent reporter) is decreased without Evi+ sEVs, there may be a dose-dependent response. Otherwise, please more clearly disclose the partial loss of Evi function without Evi+ sEVs or state the intact function of Evi without sEVs as speculative.

    We agree that Wg is likely to be reaching the muscle in the absence of Evi exosomes via conventional secretory mechanisms, and have conducted new experiments to test this hypothesis (Fig. 5). In Drosophila muscles, Wg does not signal via a conventional b-catenin pathway. Instead, neuronally-derived Wg activates cleavage of its receptor Fz2, resulting in translocation of a Fz2 C-terminal fragment into the nucleus (Mathew et al., 2005; Mosca and Schwarz, 2010). We did attempt to directly measure Wg (using antibodies or knockins) and though we were able to detect a specific presynaptic signal, the background noise throughout the postsynaptic muscle was too high for a sensible quantification. In response to the reviewer’s question and also R2.6), we collaborated with the laboratory of Timothy Mosca to test Fz2 nuclear import in Tsg101 and Hrs mutants (new Figure 5F-G). Strikingly, we found that Hrs mutants, despite being extremely sickly, have normal nuclear import of Frizzled. We also confirmed that Hrs mutants have dramatically depleted levels of all EV cargoes examined, including Evi (Figure 2A-F). On the other hand we found that Tsg101 knockdowns have dramatically reduced Wg signaling (and a concomitant defect in postsynaptic development). We do not rule out (but think it is unlikely) that very small amounts of EVs could be present in hrs but not tsg101 mutants. A more parsimonious interpretation is that additional membrane trafficking defects in the Tsg101 mutants (which are beyond the scope of this study to explore in detail) block an alternative mode of Wg release, perhaps conventional secretion. The fact that Hrs mutants, despite showing similar depletion of Evi EVs, do not have a signaling defect strongly argues that EV release per se is not required for Wg signaling.

    R1.6 To support the authors' hypothesis that Syt4 transmission via EVs is a proteostatic mechanism, the authors should determine whether Syt4 cargo localizes to lysosomal compartments in muscle, glia, or both. Otherwise, the proteostatic degradation of Syt4 via EVs is speculative.

    Our data suggest that EVs serve as one of several parallel proteostatic mechanisms for presynaptic cargoes. We have added new data to the manuscript to emphasize the advance our work makes in our understanding of these mechanisms, and have emphasized this in the discussion on p 11-12, lines 46-5).

    1. Degradation of neuronally derived EVs in glia and muscles. Previous work has shown that EV cargoes such as Evi can be found in compartments in the muscle cytoplasm, and that a-HRP-positive puncta are taken up and degraded by glial and muscle phagocytosis (Fuentes-Medel et al., 2009). These a-HRP-positive structures, despite colocalizing with EV cargoes Syt4, Nrg and APP (Walsh et al., 2021), were not previously connected to EVs. We have added new data showing that muscle or glial-specific RNAi of the phagocytic receptor Draper leads to the accumulation of EVs containing Syt4 (new Figure 7G-H)). Together with our finding (Figure 7A-F) that Syt4 is not significantly detected in the muscle cytoplasm, these results indicate that the main destination for transynaptic transfer is phagocytosis by the recipient cell. We have not been able to convincingly detect EV cargoes in the endolysosomal system of muscles, even in mutants disrupting lysosomal traffic, likely because the small number of EVs released by neurons (even over days of development) are drastically diluted in the much larger muscle cell.
    2. __Compensatory endosomophagy in the neuron. __When EV release is blocked in Hrs or Tsg101 mutants, we observe an induction of autophagy in the neuron (Figure 3B, E-G). However, in the absence of ESCRT manipulation, autophagy mutants do not accumulate EVs (Figure 3C,D. S2H-I). This suggests that autophagy is a compensatory mechanism that is induced in the absence of EV release.
    3. Retrograde transport to cell bodies: We previously found that disruption of neuronal dynactin leads to accumulation EV cargoes in presynaptic terminals (Blanchette et al., 2022), suggesting that retrograde transport is a mechanism for removal of these cargoes from synapses. Interestingly, EV release is not increased in these conditions, indicating that the retrogradely transported compartment represents a late endosome without ILVs, or an MVB that cannot fuse with the plasma membrane.

    R1.7 Please discuss alternate modes of cargo transfer from the presynaptic compartment to the postsynaptic compartment that may be utilized when EV-mediated transfer is abolished (i.e., cytonemes or tunneling nanotubules).

    We have added these possibilities to the discussion (p11 line 31), though we note that we do not observe any such structures, or indeed any Syt4 in the muscle cytoplasm, and there is no current evidence for such transsynaptic structures in this system. Conventional secretion of Wg into the extracellular space and signaling through its transmembrane receptor Frizzled2 can account for Wg signaling in the absence of exosomes.

    R1.8 OPTIONAL: Investigate the mechanism of Syt4+ sEV fusion with the postsynaptic compartment (direct fusion with the plasma membrane, receptor-mediated fusion, endocytosis and unpacking, or endocytosis and degradation).

    We note that the Budnik lab has already shown that HRP-positive EVs released by NMJs are taken up by glia and muscles (Fuentes-Medel et al., 2009), and we have added data showing that this also applies for Syt4 (Fig. 7). Our data are not consistent with Syt4 fusing with recipient cell membranes or entering the muscle cytoplasm. Further investigation of this mechanism is beyond the scope of this project.

    Given that several fundamental questions have yet to be answered regarding the biogenesis pathways and machinery utilized for EV-mediated cargo secretion, and the necessity for further TEM studies and/or work with primary cultures to characterize ILVs and EVs, >6 months is estimated to perform the necessary experiments that may require learning/ optimizing new systems.

    Minor comments:

    R1.9 Please clarify the choice of using Tsg101 KD in place of mutants of other ESCRT machinery (i.e., Hrs). Especially as when the Tsg101 mutant was characterized, you found major defects in autophagic flux that were not present for HrsD28/Df.

    Tsg101 RNAi was selected since it provides a neuron-autonomous knockdown, eliminating the complications of mutant effects in other tissues. These animals are also relatively healthy as third instar larvae compared to genomic mutants tsg1012 (L1 lethal) and HrsD28 or motor-neuron driven Vps4DN (where L3 larvae are rare). This made it easier to recover enough larvae to properly power experiments, and alleviated concerns that general sickness is contributing to the phenotype (though note that neuronal Tsg101KD does result in pupal lethality). Finally, we were unable to effectively knock down Hrs by RNAi (see R1.1). To extend our studies beyond Tsg101, we have included additional experiments in the revised manuscript showing that HrsD28 animals, despite being quite unhealthy, still retain Syt4-dependent functional plasticity (See R2.5 and R3.4) and Wg signaling.

    R1.10 Please clarify why the specific method in experiment in Fig. 4E-J was chosen. As Syt4 is a transmembrane protein, is likely undergoes degradation via the lysosome, like other membrane-bound proteins. Is it known whether the proteasome-directed nanobody is sufficient to pull Syt4 from membrane-bound compartments to undergo degradation in the proteasome? Would it make more sense to use a lysosome-directed nanobody?

    The GFP tag on Syt4 is cytosolic rather than lumenal. Our data show that when we express the proteosome-directed nanobody presynaptically, it efficiently degrades membrane-associated Syt4-GFP (Fig. 7B). Therefore we expect that this tool should be similarly effective on membrane-associated Syt4-GFP if it were exposed to the muscle cytoplasm. We have confirmed that it is effective in the muscle against DLG-GFP (Fig. S5A)

    R1.11 Please provide further methodological information regarding the sample preparation for live imaging of axons to generate kymographs found in Fig. S3.

    Additional details have been provided on p14 lines 10-24 and p15 lines 31-37.

    R1.12 In Figure 1I and 1J, include representative image and quantification of Syt4-GFP pre- and post-synaptic intensity for HrsD28/Df for consistency with ShrubKD and Vps4DN in Figure 1K-P.

    We generated and tested HrsD28; Syt4-GFP (Fig 2A,D), and HrsD28; Evi-GFP strains (Fig 2B-E). All EV cargoes exhibited a dramatic post-synaptic depletion in Hrs mutants, similar to the other ESCRT manipulations.

    R1.13 In Figure 2H, please provide a cell type marker or HRP mask with a merged image for image clarity.

    This image shows neuronal cell bodies in the ventral ganglion, which are densely packed relative to each other. The cell type specificity is provided by the motor neuron driver. We did not use a cell type marker or individually mask cells for analysis, but instead quantified intensity over the whole field of view. We can manually trace cell bodies in this image if requested, but it would not represent our ROI for analysis.

    R1.14 In Figure 4B, please provide quantification for the differences between 1) WT Mock and Tsg101 MOCK and 2) WT Stim and Tsg101KD Stim to show that upon stimulation, WT and Tsg101 undergo the same increase in the number of ghost boutons/ NMJ in Muscle 4.

    We have added these statistical comparisons to the graph (Fig. 6B)

    R1.15 In Figure 3 G and H, use consistent scale bars to compare between temperatures.

    We have removed the Shrub data at 20º as it did not provide additional insight to the manuscript.

    Reviewer #1 (Significance (Required)):

    General assessment (Strengths):

    -Use of Drosophila NMJ model system consistent with others in the field and exceptional harnessing of genetic tools for mutations across the ESCRT pathway (-0, -I, -III, etc.) -Identification of ESCRT pathway mutants that do not deplete pre-synaptic cargo levels but generate endosomal dysfunction, indicative of a possible decrease in secretion of cargoes via EVs -Implementing functional characterization of Evi/ Wg and Syt4 cargoes, consistent with previous work in the field; highly reproducible

    -Sufficiently thorough investigation of the cross-regulation of autophagy and EV biogenesis by Tsg101

    General assessment (Weaknesses):

    -Lack of investigation of known ESCRT-independent pathways/ genes involved in the generation of sEVs (i.e., nSMase2/ Ceramide) especially as it is unknown if minor sources of cargo+ EVs are sufficient in maintaining functional phenotype

    See R1.3 for comments on this point

    -Lack of sEV characterization and validation of EVs derived from mutant

    We have added STED data to measure EV size, and described the challenges in EV membrane measurements by EM in the in vivo system.

    -Does not show the loss of cargoes of interest on EVs from mutants other than through back-up of cargoes in the presynaptic endocytic pathway (Rab7, Rab5, Rab11)

    We strongly disagree with this comment. We have explicitly measured the loss of numerous cargoes in postsynaptic structures that have been rigorously established to be EVs in this and previous publications. Our findings are not limited to back-up of presynaptic structures.

    -Lack of rigorous investigation of the claim that Evi and Syt4 are released via EVs for proteostatic means is missing. Authors should demonstrate the degradation of EV cargoes by recipient cells (either muscle OR glia)

    We have added new data and discussion on multiple and compensatory proteostatic pathways.

    -If EV-mediated cargo transfer is not required, authors should investigate alternate modes of cargo transfer more rigorously (i.e., diffusion of Wg, suggest/ test hypotheses for mechanism of Syt4 function or transfer).

    We have included discussion of alternate modes of transfer for Wg (i.e. conventional secretion). By contrast, for Syt4 we believe it is acting in the donor cell without transfer, and have included alternate interpretations of the previous literature that had suggested its function in muscles.

    Advance: -Compared with other recent in vivo studies of EVs where donor EVs are loaded with a cargo, such as Cre, which uniquely identifies recipient cells through Cre recombination-mediated expression of a fluorescent reporter (Zomer et al 2015, Cell), this study relies on the readout of fluorescently tagged cargo in the recipient cells to represent transfer via EVs. While numerous studies in the Drosophila field focus on the same small set of known EV cargoes at the NMJ (Koles et al., 2012; Gross et al., 2012; Korkut et al., 2013; Korkut et al., 2009; Walsh et al., 2021), there is a noticeable lack of EV characterization based on MISEV (i.e. TEM of EVs, size distribution, enrichment of well-known EV markers [https://doi.org/10.1080/20013078.2018.1535750]) that would significantly strengthen the work and make it more widely accepted in the EV field.

    As mentioned above, many of these criteria (including EV size and enrichment of known EV markers) are already established in the previous literature for this system. As requested, we have also added similar data to our revised manuscript.

    -In this study, the use of ESCRT machinery mutants is proven as a new technical method in delineating the role of EV cargoes in cell-autonomous versus EV-dependent functions. This is the first study, to my knowledge, that has leveraged mutants from both early and late ESCRT complexes for the study of EVs in Drosophila. Additionally, the finding that some cargoes may be able to carry out their signaling functions, independent of transfer via EVs, provides key mechanistic insight into one possible role of EVs as proteostatic shuttles for cargo. This work also begins to address a fundamental question in the field, which is to delineate roles that EVs actually carry out in physiological conditions, compared to the many roles that have been shown possible in vitro.

    We appreciate the reviewer’s insight into the impact of our work.

    Audience: -Basic research (endosomal biology, ESCRT pathway, cell signaling, neurodevelopment)

    -Specialized (Drosophila, Neurobiology; Extracellular Vesicles)

    -This article will be of interest to basic scientists in the field of endosomal trafficking and extracellular vesicle biology as well as though studying the nervous system in Drosophila melanogaster. As the field of extracellular vesicle biology has broad implications in the spread of pathogenic cargoes in cancer and neurodegenerative disease, the basic biology associated with EVs has some translational relevance.

    Expertise (Keywords):

    -ESCRT and nSMase2 EV biogenesis pathways

    -EV characterization in vitro/ live imaging studies

    -EV release and uptake

    -Neuronal and glial cell biology

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This manuscript addresses the role of exosome secretion in neuromuscular junction development in Drosophila, a system that has been proposed to depend on exosomes. In particular, delivery of Wingless via exosomes has been proposed to promote structural organization of the synapse. Previously, however, the studies that proposed this model targeted the cargoes themselves, rather than targeting exosome biogenesis or secretion. In this new study, exosome biogenesis is targeted via knockdown of the ESCRT components Hrs, TSG101, and Chmp4. The authors find that some previously ascribed functions are not inhibited by these knockdowns. In particular, formation of active zones, as defined by BRP-positive puncta (total and per micrometer), and total bouton numbers. It does look like there is a partial defect in BRP-positive puncta per micrometer, but it is not significant. For ghost bouton formation, there is a similar increase in evi-mutant and ESCRT-KD NMJs (with some subtle differences depending on abdominal segment and temperature). They also examine the role of Syt4, which has been proposed to be transferred from nerve to muscle cells at the junction and to regulate mEJP frequency after stimulation. They found no difference in mEJP frequency after stimulation between WT and TSG101-KD animals, although they did not have a positive control with inhibition of Syt4. They did do an elegant experiment to demonstrate that most of extracellularly transferred Syt4 does not reach the muscle cytoplasm. Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD. There are also a couple of experiments that would enhance the manuscript. Some specific suggestions are below:

    R2.1 Title: "ESCRT disruption provides evidence against signaling functions for synaptic exosomes" seems a bit broad -- only evi/Wg and Syt4 functions were examined at NMJ synapses, not all signaling functions of all exosomes at all synapses. Something like, "ESCRT disruption provides evidence against signaling functions for exosome-carried evi/Wg and Syt4 at the neuromuscular junction" seems a bit more reasonable.

    We are open to changing the title to: “ESCRT disruption provides evidence against transsynaptic signaling functions for some extracellular vesicle cargoes” though we prefer to leave it as is since “provides evidence against” is already fairly understated.

    __ __R2.2 Abstract: the description of the actual data is very little, just one sentence saying that "many" of the signaling functions are retained with ESCRT depletion. I think a bit more focus on the actual data is warranted.

    We have edited the abstract to include more detail on the signaling phenotypes.

    __

    __R2.3 Results section:

    Fig 3: What does A2 and A3 mean for the graphs in c,d,e, g, h? Please specify in figure legend.

    We have described in the figure legends that A2 and A3 refer to specific abdominal segments in the larvae.

    R2.4 The sentence "Further, active zones in Tsg101KD appeared morphologically normal by TEM (Fig.2B)." is confusing to me. What do you mean by that? Are you referring to the following two sentences about feathery DLG and SSR? But the feathery DLG I presume is in Fig 3, where that staining is. And I also don't know what feathery DLG means -- it should be pointed out in the appropriate image.

    Presynaptic active zones are defined by an electron-dense T-shaped pedestal at sites of synaptic vesicle release, and can be seen in the TEM in what is now Figure 3B, marked as AZ. We have also labeled AZ by immunofluorescence (Fig. 5A) and they appear normal.

    By contrast, Dlg primarily labels the postsynaptic apparatus associated with the infoldings of the muscle membrane. In control animals, Dlg immunostaining is relatively tightly and smoothly clustered within ~1µm of the presynaptic neuron. By contrast, in Evi mutants, there are wisps of Dlg-positive structures extending from the bouton periphery. We have added arrows in what is now Fig. 5C to indicate the feathery structures.

    R2.5 Fig 4 addresses Syt4 function. However, there is no positive control inhibiting Syt4 to see if there is a change. Just comparison of WT and TSG101. It seems like this positive control is in order.

    We have added the positive control (Fig. 6E-F) reproducing the previously reported result that Syt4 mutants lack the high-frequency stimulation-induced increase in mEPSP frequency (HFMR). We have also added new data on HrsD28 genomic mutants. Despite the fact that few of these larvae survive and they are quite unhealthy, they still exhibit robust HFMR, similar to the Tsg101KD larvae, strongly supporting our hypothesis.

    R2.6 Discussion: I think some discussion of what ghost boutons are and what the possible significance is of the evi and ESCRT mutant phenotype of enhanced ghost bouton formation

    We have added more discussion on the ghost bouton phenotype (p11 lines 5-14), especially in light of our new findings that Hrs and Tsg101 mutants may distinguish alternative modes of Wg secretion (see R1.5)

    R2.7 Also, in the Discussion, it is mentioned that Wg probably gets secreted in the ESCRT mutants -- presumably this accounts for the discrepancy between evi mutants and the ESCRT mutants. An experiment to actually test this would greatly enhance the manuscript.

    We have added this experiment as addressed in R1.5

    Reviewer #2 (Significance (Required)):

    Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Dresselhaus et al. investigates signaling functions for synaptic exosomes at the Drosophila NMJ. Exosomes are widely seen in vivo and in vitro. They are clearly sufficient to induce signaling responses in vitro, but whether they normally fulfill signaling functions in vivo has not been rigorously addressed. The authors make use of several mutants that block exosome release to test whether exosome release is important for two distinct signaling pathways: the Evi/Wg pathway and the Syt4 signaling pathway. Both pathways have been implicated in neuron to muscle signaling. Surprisingly, the authors find scant evidence that exosome release is required for either pathway. They convincingly show that knockdown of Tsg101 (an ESCRT-I component) does not phenocopy many synaptic phenotypes of either wg or syt4. Instead, they propose that in vivo, exosomes may serve as a proteostatic mechanism, as a mechanism for the neuron to dispose of unwanted/damaged proteins.

    Specific comments are below:

    R3.1 Loss of Tsg101 has been linked to upregulated MAPK stress signaling pathways and autophagy. Thus, it's possible that activating such compensatory mechanisms in Tsg101 knockdown animals could mask phenotypes associated with specific loss of EV cargoes such as Wg or Syt4. Indeed, the authors demonstrate that loss of Tsg101 and Hrs have very different effects on synaptic autophagy. To provide additional evidence that Wg or Syt4 signaling is independent of EV release, it would be good to check for wg/syt4 phenocopy in additional ESCRT complex mutants. I understand they did a bit with Shrub knockdown at low temperature in Figure 3, but the temperature-dependence of the ghost bouton phenotype clouds the interpretation. Could the authors try a motorneuron driver with a more restricted phenotype to overcome the lethality issues, or alternatively use one of their other ESCRT component mutants? This is obviously the central claim of the manuscript, and it would be strengthened by carrying out phenotypic analysis in mutants other than the Tsg101 RNAi line.

    As noted for R2.5, we have added HFMR experiments for the HrsD28 genomic mutant, and found that despite being very unhealthy, they exhibit robust HFMR similar to Tsg101KD. We also confirmed dramatic depletion of Syt4 EVs in the HrsD28 mutant. Thus, the preserved Syt4 signaling function in ESCRT mutants with depleted EV Syt4 is not restricted to Tsg101, and does not depend on the co-occurring autophagy phenotype.

    R3.2 In Figure 1, the authors show that neuronal Tsg101 RNAi dramatically reduces "postsynaptic" levels of exosome cargoes at the L3 stage to argue that exosome release is blocked in this mutant. While this seems very likely at the L3 stage, it is unclear when Tsg101 levels are reduced and thus when exosome release is impaired in this background. This is important because we don't know when these signaling pathways act. For example, it is possible that the critical period for Wg and Syt4 signaling is during the L1 stage, and that Tsg101 knockdown is incomplete at that stage. It is important to assay exosome release at earlier larval stage, particularly when RNAi is the method used to reduce gene function.

    We have conducted this experiment. We noted accumulation of cargoes in Tsg101KD L1 larvae, indicating that the RNAi is effective early in development. However, we do not find many EVs in either wild-type or Tsg101KD first instar larvae (red is a-HRP, green is Syt4-GFP). This argues that it is unlikely that EV-mediated signaling has a critical period earlier in development. It is likely that the accumulation of EVs that we observe trapped in the muscle membrane reticulum in third instar larvae were laid down over days or hours of development. We do not propose to include these data in the manuscript unless the editors and reviewers prefer that we do so.

    R3.3 If the Syt4 and Evi exosomes do not serve major signaling roles and are in fact neuronal waste, it seems likely they are phagocytosed by glia. Are levels of non-neuronal Syt4/Evi levels increased when glial phagocytosis in blocked (eg in draper mutants)?

    As mentioned above, the Budnik lab previously showed that uptake and degradation of postsynaptic a-HRP-positive structures depends on glial and muscle phagocytosis.a-HRP recognizes a number of neuronally-derived glycoproteins (Snow et al., 1987). Though the Budnik lab had not previously linked these structures to EVs, we do know that they very strongly colocalize with known EV cargoes and depend on the exact same membrane traffic machinery for release, arguing that some a-HRP antigen proteins are also EV cargoes (Blanchette et al., 2022). To close this loop. we have added data showing that Syt4-positive EVs also depend on Draper for their clearance (Fig 7).

    R3.4 For the HFMR experiment, it would be good to see the syt4-dependent phenotype as a positive control.__ __

    As mentioned for R2.5, we have added the Syt4 positive control (Figure 6E,F), which fails to show HFMR as expected.

    .__ __R3.5 In the abstract, the authors state that, "the cargoes are likely to function cell autonomously in the motorneuron". Isn't it alternatively possible that these proteins (wg in particular) could signal to the muscle in a non-exosome dependent pathway?

    Yes, we believe that Wg is likely released by another mechanism (perhaps conventional secretion). As noted for R1.5 and R2.6, we have added new data in Fig. 5 showing that Frizzled nuclear import IS NOT disrupted in Hrs mutants, despite dramatic loss of Evi EVs. Interestingly Frizzled nuclear import (and postsynaptic development) IS altered in neuronal Tsg101KD larvae, which disrupt additional membrane trafficking pathways beyond EV release (see Fig. 3). This is particularly interesting in light of the normal Syt4 signaling in Tsg101KD larvae, and supports the hypothesis that Syt4 can function without leaving the neuron, while Wg must be released, albeit not via Hrs-dependent EV formation. Another (less parsimonious) interpretation is that very small amounts of Wg release in the Hrs mutant are sufficient to promote Frizzled nuclear import.

    Reviewer #3 (Significance (Required)):

    This is an important paper that is well-organized and logically presented. It makes a clear and largely compelling case against major signaling roles for exosomes at this synapse. The authors should be commended for publishing this work, which demands a re-evaluation of proposed key roles for exosomes at the fly NMJ. Given the intense interest in exosomes in neurobiology, this paper will be of great interest to neuronal cell biologists working across systems.

    We thank the reviewer for their appreciation of the impact of our work on the field.

    Back, M.J., H.C. Ha, Z. Fu, J.M. Choi, Y. Piao, J.H. Won, J.M. Jang, I.C. Shin, and D.K. Kim. 2018. Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide. Cell Death Dis. 9:670.

    Blanchette, C.R., and A.A. Rodal. 2020. Mechanisms for biogenesis and release of neuronal extracellular vesicles. Curr Opin Neurobiol. 63:104-110.

    Blanchette, C.R., A.L. Scalera, K.P. Harris, Z. Zhao, E.C. Dresselhaus, K. Koles, A. Yeh, J.K. Apiki, B.A. Stewart, and A.A. Rodal. 2022. Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery. J. Cell Biol. 10.1083/jcb.202112094.

    Choezom, D., and J.C. Gross. 2022. Neutral sphingomyelinase 2 controls exosome secretion by counteracting V-ATPase-mediated endosome acidification. J Cell Sci. 135.

    Enneking, E.M., S.R. Kudumala, E. Moreno, R. Stephan, J. Boerner, T.A. Godenschwege, and J. Pielage. 2013. Transsynaptic coordination of synaptic growth, function, and stability by the L1-type CAM Neuroglian. PLoS Biol. 11:e1001537.

    Fuentes-Medel, Y., M.A. Logan, J. Ashley, B. Ataman, V. Budnik, and M.R. Freeman. 2009. Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris. PLoS Biol. 7:e1000184.

    Koles, K., J. Nunnari, C. Korkut, R. Barria, C. Brewer, Y. Li, J. Leszyk, B. Zhang, and V. Budnik. 2012. Mechanism of evenness interrupted (Evi)-exosome release at synaptic boutons. J Biol Chem. 287:16820-16834.

    Korkut, C., B. Ataman, P. Ramachandran, J. Ashley, R. Barria, N. Gherbesi, and V. Budnik. 2009. Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell. 139:393-404.

    Korkut, C., Y. Li, K. Koles, C. Brewer, J. Ashley, M. Yoshihara, and V. Budnik. 2013. Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron. 77:1039-1046.

    Lauwers, E., Y.C. Wang, R. Gallardo, R. Van der Kant, E. Michiels, J. Swerts, P. Baatsen, S.S. Zaiter, S.R. McAlpine, N.V. Gounko, F. Rousseau, J. Schymkowitz, and P. Verstreken. 2018. Hsp90 Mediates Membrane Deformation and Exosome Release. Mol Cell. 71:689-702 e689.

    Mathew, D., B. Ataman, J. Chen, Y. Zhang, S. Cumberledge, and V. Budnik. 2005. Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2. Science. 310:1344-1347.

    Moberg, K.H., S. Schelble, S.K. Burdick, and I.K. Hariharan. 2005. Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev Cell. 9:699-710.

    Mosca, T.J., and T.L. Schwarz. 2010. The nuclear import of Frizzled2-C by Importins-beta11 and alpha2 promotes postsynaptic development. Nat Neurosci. 13:935-943.

    Niekamp, P., F. Scharte, T. Sokoya, L. Vittadello, Y. Kim, Y. Deng, E. Sudhoff, A. Hilderink, M. Imlau, C.J. Clarke, M. Hensel, C.G. Burd, and J.C.M. Holthuis. 2022. Ca(2+)-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair. Nat Commun. 13:1875.

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  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Dresselhaus et al. investigates signaling functions for synaptic exosomes at the Drosophila NMJ. Exosomes are widely seen in vivo and in vitro. They are clearly sufficient to induce signaling responses in vitro, but whether they normally fulfill signaling functions in vivo has not been rigorously addressed. The authors make use of several mutants that block exosome release to test whether exosome release is important for two distinct signaling pathways: the Evi/Wg pathway and the Syt4 signaling pathway. Both pathways have been implicated in neuron to muscle signaling. Surprisingly, the authors find scant evidence that exosome release is required for either pathway. They convincingly show that knockdown of Tsg101 (an ESCRT-I component) does not phenocopy many synaptic phenotypes of either wg or syt4. Instead, they propose that in vivo, exosomes may serve as a proteostatic mechanism, as a mechanism for the neuron to dispose of unwanted/damaged proteins.

    Specific comments are below:

    Loss of Tsg101 has been linked to upregulated MAPK stress signaling pathways and autophagy. Thus, it's possible that activating such compensatory mechanisms in Tsg101 knockdown animals could mask phenotypes associated with specific loss of EV cargoes such as Wg or Syt4. Indeed, the authors demonstrate that loss of Tsg101 and Hrs have very different effects on synaptic autophagy. To provide additional evidence that Wg or Syt4 signaling is independent of EV release, it would be good to check for wg/syt4 phenocopy in additional ESCRT complex mutants. I understand they did a bit with Shrub knockdown at low temperature in Figure 3, but the temperature-dependence of the ghost bouton phenotype clouds the interpretation. Could the authors try a motorneuron driver with a more restricted phenotype to overcome the lethality issues, or alternatively use one of their other ESCRT component mutants? This is obviously the central claim of the manuscript, and it would be strengthened by carrying out phenotypic analysis in mutants other than the Tsg101 RNAi line.

    In Figure 1, the authors show that neuronal Tsg101 RNAi dramatically reduces "postsynaptic" levels of exosome cargoes at the L3 stage to argue that exosome release is blocked in this mutant. While this seems very likely at the L3 stage, it is unclear when Tsg101 levels are reduced and thus when exosome release is impaired in this background. This is important because we don't know when these signaling pathways act. For example, it is possible that the critical period for Wg and Syt4 signaling is during the L1 stage, and that Tsg101 knockdown is incomplete at that stage. It is important to assay exosome release at earlier larval stage, particularly when RNAi is the method used to reduce gene function.

    If the Syt4 and Evi exosomes do not serve major signaling roles and are in fact neuronal waste, it seems likely they are phagocytosed by glia. Are levels of non-neuronal Syt4/Evi levels increased when glial phagocytosis in blocked (eg in draper mutants)?

    For the HFMR experiment, it would be good to see the syt4-dependent phenotype as a positive control.

    In the abstract, the authors state that, "the cargoes are likely to function cell autonomously in the motorneuron". Isn't it alternatively possible that these proteins (wg in particular) could signal to the muscle in a non-exosome dependent pathway?

    Significance

    This is an important paper that is well-organized and logically presented. It makes a clear and largely compelling case against major signaling roles for exosomes at this synapse. The authors should be commended for publishing this work, which demands a re-evaluation of proposed key roles for exosomes at the fly NMJ. Given the intense interest in exosomes in neurobiology, this paper will be of great interest to neuronal cell biologists working across systems.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript addresses the role of exosome secretion in neuromuscular junction development in Drosophila, a system that has been proposed to depend on exosomes. In particular, delivery of Wingless via exosomes has been proposed to promote structural organization of the synapse. Previously, however, the studies that proposed this model targeted the cargoes themselves, rather than targeting exosome biogenesis or secretion. In this new study, exosome biogenesis is targeted via knockdown of the ESCRT components Hrs, TSG101, and Chmp4. The authors find that some previously ascribed functions are not inhibited by these knockdowns. In particular, formation of active zones, as defined by BRP-positive puncta (total and per micrometer), and total bouton numbers. It does look like there is a partial defect in BRP-positive puncta per micrometer, but it is not significant. For ghost bouton formation, there is a similar increase in evi-mutant and ESCRT-KD NMJs (with some subtle differences depending on abdominal segment and temperature). They also examine the role of Syt4, which has been proposed to be transferred from nerve to muscle cells at the junction and to regulate mEJP frequency after stimulation. They found no difference in mEJP frequency after stimulation between WT and TSG101-KD animals, although they did not have a positive control with inhibition of Syt4. They did do an elegant experiment to demonstrate that most of extracellularly transferred Syt4 does not reach the muscle cytoplasm. Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD. There are also a couple of experiments that would enhance the manuscript. Some specific suggestions are below:

    Title: "ESCRT disruption provides evidence against signaling functions for synaptic exosomes" seems a bit broad -- only evi/Wg and Syt4 functions were examined at NMJ synapses, not all signaling functions of all exosomes at all synapses. Something like, "ESCRT disruption provides evidence against signaling functions for exosome-carried evi/Wg and Syt4 at the neuromuscular junction" seems a bit more reasonable.

    Abstract: the description of the actual data is very little, just one sentence saying that "many" of the signaling functions are retained with ESCRT depletion. I think a bit more focus on the actual data is warranted.

    Results section: Fig 3: What does A2 and A3 mean for the graphs in c,d,e, g, h? Please specify in figure legend.

    The sentence "Further, active zones in Tsg101KD appeared morphologically normal by TEM (Fig. 2B)." is confusing to me. What do you mean by that? Are you referring to the following two sentences about feathery DLG and SSR? But the feathery DLG I presume is in Fig 3, where that staining is. And I also don't know what feathery DLG means -- it should be pointed out in the appropriate image.

    Fig 4 addresses Syt4 function. However, there is no positive control inhibiting Syt4 to see if there is a change. Just comparison of WT and TSG101. It seems like this positive control is in order. Discussion: I think some discussion of what ghost boutons are and what the possible significance is of the evi and ESCRT mutant phenotype of enhanced ghost bouton formation

    Also, in the Discussion, it is mentioned that Wg probably gets secreted in the ESCRT mutants -- presumably this accounts for the discrepancy between evi mutants and the ESCRT mutants. An experiment to actually test this would greatly enhance the manuscript.

    Significance

    Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this paper, Dresselhaus et al (2023) investigate the possibility that known cargoes of extracellular vesicles (EVs) released at the Drosophila neuromuscular junction have cell-autonomous functions rather than functions specifically conferred as a condition of their release in EVs, in vivo. To do so, authors focus their studies on use of Tsg101-KD, a mutant of the ESCRT-I machinery, of the ESCRT EV biogenesis pathway, and are able to show that for some endogenously-expressed, fluorescently-tagged cargoes, fluorescence intensity in the pre-synaptic compartment is significantly elevated (Syt4 and Evi) and the postsynaptic intensity in the muscle is significantly decreased (Syt4, Evi, APP, and Nrg). These findings suggest that these cargoes become trapped in the endosomal system (colocalizing with early, late, and recycling endosomal compartments), rather than undergoing secretion in EVs targeting post-synaptic muscle and glia as usual. This phenotype is recapitulated for select cargoes using mutants of both early and late components of ESCRT pathway machinery. They further characterize the Tsg101 mutant, demonstrating co-occurrence of an autophagic flux defect , but as the cargo phenotype is present without induction of the autophagic flux defect for their Hrs mutants, authors suggest the overlapping role of Tsg101 in autophagy is independent of its role in the ESCRT pathway/ EV secretion. Subsequently, they use previously defined functional phenotypes of the Evi (number of active zones, number of boutons, number of developmentally-arrested ghost boutons) and Syt-4 (number of transient ghost boutons and mEJPs) cargoes to show a minimal dependence on cargo delivery via ESCRT-derived EVs for these cargoes to carry out their synaptic growth and plasticity functions in vivo. However, it should be notes that for Evi/ Wg cargo, there is a slight increase in developmentally-arrested ghost boutons suggesting the cargo may not be entirely independent of EV-mediated cargo delivery. Finally, authors express an anti-GFP proteasome-directed nanobody using motor neuron or muscle-specific drivers and find that Syt4-GFP cargo doesn't enter muscle cytoplasm as fluorescence is maintained and cargo is not degraded by the muscle proteasome. While authors suggest this as evidence of EV-mediated transfer for cargo proteostasis, it is not explicitly shown that Syt4 cargo is, in fact, trafficked and degraded by the lysosome or hypothesized how Syt4 function or post-synaptic localization may be carried out independently of EVs.

    Major comments:

    • It is difficult to evaluate the findings of this study without knowing the extent of ESCRT pathway impairment. Please provide data quantifying the degree of knockdown/ mutant expression for each ESCRT component (i.e., western blot)
    • Loss of ESCRT machinery likely disrupts the release of small EVs to a significant extent; however, the authors do not show that EV release is entirely lost, only that 1) cargoes are backed up in the endosomal system due to endosomal dysfunction and 2) fluorescence of cargoes in the postsynaptic compartment is diminished. To claim that ESCRT-derived EVs with the relevant cargoes are lost, the authors should perform immunogold labelling with TEM. This would provide direct evidence that the cargoes examined here are packaged in ILVs, and that the ILVs are of a size (~50-150nm) consistent with exosomes (which should really be referred to as small extracellular vesicles (sEVs) per the minimal information for studies of extracellular vesicles (MISEV 2018 [https://doi.org/10.1080/20013078.2018.1535750])
    • Additionally, EM would show the loss of cargo packaging and provide information about where these cargoes localize in the presence of ESCRT mutants/loss-of-function.
    • Other biogenesis pathways utilize multivesicular bodies to generate EVs, most prominently the nSMase2/ceramide synthesis pathway (which operates in an ESCRT-independent manner). It is possible that this pathway compensates when there are defects in the canonical ESCRT pathway. Thus, it is imperative for the authors to show that the cargo secretion no longer occurs in the presence of ESCRT mutations/loss-of-function. The authors should also use nSMase2 pathway mutants to see if the phenotypes in cargo trafficking (i.e., pre/ post-synaptic protein levels) are recapitulated.
    • The authors' findings support that cargo trafficking is affected by widespread endosomal dysfunction but doesn't cleanly prove that 1) synaptic sEV release is lost and 2) that cargo-specific sEVs are lost. As previously mentioned, loss of cargo+ ILVs in MVEs by TEM could demonstrate this, but another useful approach would be to include in vitro Drosophila primary neuronal culture/ EV isolation and mass spec/proteomic characterization studies as proof of concept. According to widely agreed upon guidelines in the EV field, the authors should directly characterize their EV population to show 1) the appropriate size distribution associated with exosomes/sEVs, 2) the presence of traditional EV markers (i.e., tetraspanins), 3) changes in overall EV count by ESCRT mutants, and 4) decreased levels of cargo(es) of interest in the presence of ESCRT mutants/loss-of-function. In vitro experiments would be particularly helpful for quantifying the degree of loss of cargo-specific EVs with each ESCRT mutant. These experiments could also investigate the possibility that cargoes are secreted in nSMase2/ Ceramide-derived EVs, by showing that EV cargo levels are unaffected in nSMase mutants.
    • During functional tests of Evi+ motor neurons lacking generation of Evi+ EVs, there is a slight defect observed, namely the increased formation of developmentally arrested ghost boutons when Evi secretion in sEVs is lost. As mentioned, Evi is a transporter of Wg and it is possible for Wg to be transmitted between cells via normal diffusion. Thus, some basal levels of Wg may be reaching the muscle when its transfer via sEVs is abolished, and these basal levels may be sufficient to phenocopy the WT in the number of active zones and boutons. Is it possible that this element of Evi/ Wg function is dose-dependent and thus reliant on the extra Evi/ Wg transferred via sEVs? If possible, the authors should use a Wnt-signaling pathway reporter (i.e., fluorescently tagged Beta-Catenin) to measure the levels of Wnt signaling activity in the muscle when Evi/Wg+ EVs are present vs. abolished. If the degree of Wnt signaling (readout would be intensity of fluorescent reporter) is decreased without Evi+ sEVs, there may be a dose-dependent response. Otherwise, please more clearly disclose the partial loss of Evi function without Evi+ sEVs or state the intact function of Evi without sEVs as speculative.
    • To support the authors' hypothesis that Syt4 transmission via EVs is a proteostatic mechanism, the authors should determine whether Syt4 cargo localizes to lysosomal compartments in muscle, glia, or both. Otherwise, the proteostatic degradation of Syt4 via EVs is speculative.
    • Please discuss alternate modes of cargo transfer from the presynaptic compartment to the postsynaptic compartment that may be utilized when EV-mediated transfer is abolished (i.e., cytonemes or tunneling nanotubules).
    • OPTIONAL: Investigate the mechanism of Syt4+ sEV fusion with the postsynaptic compartment (direct fusion with the plasma membrane, receptor-mediated fusion, endocytosis and unpacking, or endocytosis and degradation).
    • Given that several fundamental questions have yet to be answered regarding the biogenesis pathways and machinery utilized for EV-mediated cargo secretion, and the necessity for further TEM studies and/or work with primary cultures to characterize ILVs and EVs, >6 months is estimated to perform the necessary experiments that may require learning/ optimizing new systems.

    Minor comments:

    • Please clarify the choice of using Tsg101 KD in place of mutants of other ESCRT machinery (i.e., Hrs). Especially as when the Tsg101 mutant was characterized, you found major defects in autophagic flux that were not present for HrsD28/Df.
    • Please clarify why the specific method in experiment in Fig. 4E-J was chosen. As Syt4 is a transmembrane protein, is likely undergoes degradation via the lysosome, like other membrane-bound proteins. Is it known whether the proteasome-directed nanobody is sufficient to pull Syt4 from membrane-bound compartments to undergo degradation in the proteasome? Would it make more sense to use a lysosome-directed nanobody?
    • Please provide further methodological information regarding the sample preparation for live imaging of axons to generate kymographs found in Fig. S3.
    • In Figure 1I and 1J, include representative image and quantification of Syt4-GFP pre- and post-synaptic intensity for HrsD28/Df for consistency with ShrubKD and Vps4DN in Figure 1K-P.
    • In Figure 2H, please provide a cell type marker or HRP mask with a merged image for image clarity.
    • In Figure 4B, please provide quantification for the differences between 1) WT Mock and Tsg101 MOCK and 2) WT Stim and Tsg101KD Stim to show that upon stimulation, WT and Tsg101 undergo the same increase in the number of ghost boutons/ NMJ in Muscle 4.
    • In Figure 3 G and H, use consistent scale bars to compare between temperatures.

    Significance

    General assessment (Strengths):

    • Use of Drosophila NMJ model system consistent with others in the field and exceptional harnessing of genetic tools for mutations across the ESCRT pathway (-0, -I, -III, etc.)
    • Identification of ESCRT pathway mutants that do not deplete pre-synaptic cargo levels but generate endosomal dysfunction, indicative of a possible decrease in secretion of cargoes via EVs
    • Implementing functional characterization of Evi/ Wg and Syt4 cargoes, consistent with previous work in the field; highly reproducible
    • Sufficiently thorough investigation of the cross-regulation of autophagy and EV biogenesis by Tsg101

    General assessment (Weaknesses):

    • Lack of investigation of known ESCRT-independent pathways/ genes involved in the generation of sEVs (i.e., nSMase2/ Ceramide) especially as it is unknown if minor sources of cargo+ EVs are sufficient in maintaining functional phenotype
    • Lack of sEV characterization and validation of EVs derived from mutant
    • Does not show the loss of cargoes of interest on EVs from mutants other than through back-up of cargoes in the presynaptic endocytic pathway (Rab7, Rab5, Rab11)
    • Lack of rigorous investigation of the claim that Evi and Syt4 are released via EVs for proteostatic means is missing. Authors should demonstrate the degradation of EV cargoes by recipient cells (either muscle OR glia)
    • If EV-mediated cargo transfer is not required, authors should investigate alternate modes of cargo transfer more rigorously (i.e., diffusion of Wg, suggest/ test hypotheses for mechanism of Syt4 function or transfer).

    Advance:

    • Compared with other recent in vivo studies of EVs where donor EVs are loaded with a cargo, such as Cre, which uniquely identifies recipient cells through Cre recombination-mediated expression of a fluorescent reporter (Zomer et al 2015, Cell), this study relies on the readout of fluorescently tagged cargo in the recipient cells to represent transfer via EVs. While numerous studies in the Drosophila field focus on the same small set of known EV cargoes at the NMJ (Koles et al., 2012; Gross et al., 2012; Korkut et al., 2013; Korkut et al., 2009; Walsh et al., 2021), there is a noticeable lack of EV characterization based on MISEV (i.e. TEM of EVs, size distribution, enrichment of well-known EV markers [https://doi.org/10.1080/20013078.2018.1535750]) that would significantly strengthen the work and make it more widely accepted in the EV field.
    • In this study, the use of ESCRT machinery mutants is proven as a new technical method in delineating the role of EV cargoes in cell-autonomous versus EV-dependent functions. This is the first study, to my knowledge, that has leveraged mutants from both early and late ESCRT complexes for the study of EVs in Drosophila. Additionally, the finding that some cargoes may be able to carry out their signaling functions, independent of transfer via EVs, provides key mechanistic insight into one possible role of EVs as proteostatic shuttles for cargo. This work also begins to address a fundamental question in the field, which is to delineate roles that EVs actually carry out in physiological conditions, compared to the many roles that have been shown possible in vitro.

    Audience:

    • Basic research (endosomal biology, ESCRT pathway, cell signaling, neurodevelopment)
    • Specialized (Drosophila, Neurobiology; Extracellular Vesicles)
    • This article will be of interest to basic scientists in the field of endosomal trafficking and extracellular vesicle biology as well as though studying the nervous system in Drosophila melanogaster. As the field of extracellular vesicle biology has broad implications in the spread of pathogenic cargoes in cancer and neurodegenerative disease, the basic biology associated with EVs has some translational relevance.

    Expertise (Keywords):

    • ESCRT and nSMase2 EV biogenesis pathways
    • EV characterization in vitro/ live imaging studies
    • EV release and uptake
    • Neuronal and glial cell biology
  5. Version published to 10.1101/2023.04.22.537920v2 on bioRxiv
  6. Version published to 10.1101/2023.04.22.537920v1 on bioRxiv